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Pouring Worm Plates (NGM)
To make NGM plates at the Buck: Cribbed from the excellent Chin-Sang lab web page at http://130.15.90.245/wormlab_recipe_book.htm , which is the first Google hit for "NGM plates recipe". We buy these plates as of 8/6/12 for worm-lifespan sized (60mm) plates: http://www.tritechresearch.com/T3306.html , and for larger chunk plates we use sterile 100X15mm BD Falcon 351029 'plates. '''Standard worm (NGM agar) plates ' '''Per 1 liter, to be autoclaved in 2l flask: Milli-Q Water 975ml (add 200ml before adding solids, then remaining 775ml after NaCl 3g P/N: EMD, SX0420-3 Bacto-Agar 17g P/N: BD, 214010 Bacto-Peptone 2.5g P/N: BD, 211677 Cholesterol 1ml of 10mg/ml stock in EtOH P/N: Bioworld, 40330036-1 So then, to make up 3 liters in a 6 liter Erlenmeyer flask: Do this by adding 1 liter milli-Q water , then all of the other ingredients and a teflon stirbar on the stir- plate, then bringing the final milli-Q water volume up to 3 liters. All of the ingredients are on the shelves above the balance, labeled, except cholesterol stock which is in a 50ml conical in the top shelf of the door of the -20C that is near the -80 and 30C shaker , also clearly labeled. Cholesterol may also be in door of 4C #3. Autoclave (Hey If you are in the Kennedy Lab for our Autoclave just press the liquid cycle button) 60 min on liquid cycle (depending on how many liters are being autoclaved), - this means bring it to Justin and tell him to bring it up to you when it comes out, and to let you know when you should expect it roughly. After autoclaving, using [[Sterile Technique|'sterile' technique]], add the following, per liter:' ' 1M CaCl2 - 1 ml 1M MgSO4 - 1ml 1M KH2PO4, pH 6 - 25ml These stocks are all above Mark's bench, or above Chris's old bench where the worm scope is located. Swirl to mix thoroughly after each addition; after all additions done, pour plates- for large plates pour by hand, for small use the eppendorf repeat pipettor with the 50ml adaptor, both above Mark's bench. Pour plates on a 70% EtOH-cleaned bench, with a burner going for vortex. To make up the stock solutions (Keep Solutions Sterile): 1 M CaCl2 To make 1 liter 110.9 g CaCl2 or 147.01 g CaCl2*2H2O P/N: (Fisher, C79-500) dH2O to 1 liter Aliquot 100 ml/bottle, autoclave 1 M MgSO4 ''' To make 1 liter 120.3 g MgSO4 or 246.47 g MgSO4*7H2O P/N: (Bioworld, 413000-40-1) dH2O to 1 liter Aliquot 100 ml/bottle, autoclave Aliquot to 250 ml bottles, autoclave on liquid cycle '''1M K phosphate buffer, pH 6.0 To make 1 liter KH2PO4 136.1 g P/N: (VWR, BDH0268-5006) KOH 17.9 g P/N: (Bioworld, 41600132-1) Dissolve in ~750 ml dH2O Check pH and adjust if necessary (it should be close) Bring volume to 1l with dH2O, aliquot to 500 ml bottle, autoclave A NOTE ON ADJUSTING PH OF SOLUTIONS: For the love of all that is holy please do not use the pH meter without getting a tutorial first; there are people who loooooooooove to have very long meetings / lectures about imagined misuse of the pH meter, and to be fair this is because it is the pH meter we currently have is a very temperamental and unfortunate design, which is very easy to break and has been broken previously more than once. If I have to hear some bullshit about the pH meter because of you I will be officially pissed. After letting the Plates Solidify (1-2 days) please mark the plate with the appropriate colors, see this page Worm Plate Color Code.